Pancreatic cancer remains the most lethal cancer known to man, owing primarily to its lack of early symptom resulting in late diagnosis and lack of consistent treatment efficacy. MUC4 is a large-sized membrane- anchored mucin which is aberrantly overexpressed by most human carcinomas. We have previously reported that MUC4 promotes pancreatic tumor growth and metastasis by multiple mechanisms. In parallel to these studies, using immunohistochemistry in tissue microarrays containing well characterized tumors tissues, we observed elevated expression of neutrophil gelatinase associated lipocalin (NGAL), also known as Lipocalin 2 or uterocalin, during pancreatic cancer progression. Interestingly, the expression of NGAL correlated with MUC4 expression in these tissues. Immunocytochemistry showed co-expression of NGAL and MUC4 in pancreatic cancer tissue sections. Recent studies by our group have shown that NGAL is a potential diagnostic biomarker and its expression is upregulated as early as the preneoplastic PanIN-2 stage. Additionally, preliminary studies have shown that, in the pancreatic cancer cell line CaPan-1, MUC4 knockdown leads to decreased expression of NGAL. In the present study, I will further investigate the link between NGAL and MUC4 in pancreatic cancer using the cell lines CoLo-357, CaPan-1, MiaPaCa, and PANC-1 as well as healthy and neoplastic pancreatic tissue from both humans and murine models in pursuit of the hypothesis that MUC4, acting through HER2, regulates NGAL expression in pancreatic cancer and that this connection will prove highly relevant to the prognostic abilities of NGAL as a pancreatic cancer surrogate biomarker. In pursuit of this investigation I have formulated three specific aims. In the first specific aim, I will use knockdown and overexpression experiments as well as Western blotting, Quantitative-Real Time PCR, and immunohistochemistry to determine if expression of NGAL and MUC4 are reliably linked in pancreatic cancer and if this link is present at the translational and/or transcriptional level. In the second aim, I will elucidate the pathway by which NGAL and MUC4 expression are linked using currently-known upstream and downstream proteins and pathways as a starting-point. Specifically, based on literature and my preliminary results, I will begin by assessing if MUC4 regulates NGAL expression through HER2. In the third aim, I will use an immunocompetent murine model genetically manipulated to spontaneously produce pancreatic cancer that has been developed in our lab to assess the correlation between tissue and plasma NGAL and tissue MUC4 levels throughout the development of and advancement of pancreatic cancer. Through these aims, I expect to conclusively elucidate the biochemical connection between MUC4 and NGAL while improving on current evidence potentiating NGAL's utility as both an early diagnostic and prognostic surrogate biomarker for pancreatic cancer. PUBLIC HEALTH RELEVANCE: Pancreatic cancer remains the most lethal human malignancy, owing mostly to a lack of early diagnosis and abysmal treatment efficacy in late-stage disease. Preliminary evidence suggests that MUC4, one of the earliest and most differentially upregulated genes in pancreatic cancer and which positively correlates with metastatic and tumorigenic potential, is upstream of NGAL, a secreted protein also found to be overexpressed in pancreatic tumor tissues as well as in the peripheral blood of pancreatic cancer patients. This study aims to define the mechanism by which MUC4 and NGAL expressions are linked, thereby bolstering the evidence for peripheral blood NGAL's use as a potential early diagnostic and prognostic marker in pancreatic cancer.